This proposal involves development of three very promising vector systems to facilitate immunization with antigens of BioDefense (BD) importance. The overall goal is to develop the expertise to rapidly construct vaccine prototypes and evaluate immune responses against expressed BD antigens. The vector systems will include safe, live vaccines based on Vibrio cholerae, killed vaccines based on Escherichia coli, and live, virus vectors based on replication-incompetent herpes simplex virus (HSV). Vaccine vectors will be tested for ability to promote antibody and cell-mediated immune responses to each of three BD antigens: the protective antigen (PA) of Bacillus anthracis, listeriolysin O (LLO) of Listeria monocytogenes, and the West Nile virus envelope protein (WNE). The proposed program will provide preclinical information needed to justify testing the antigen-vector constructs for safety and immunogenicity in human volunteers. For bacterial vectors, BD antigen constructs will be introduced as chromosomal insertions by transposition or by directed homologous recombination. Expression profiling using genomic microarrays will guide design of vectors for optimal constitutive or in vivo induced BD antigen expression. E. coli K-12 engineered to express cytoplasmic LLO will be used to deliver BD antigens to the MHC Class I pathway of antigen processing and presentation. Technology will also be developed for targeting E. coli and V. cholerae-based vectors into nonprofessional antigen presenting cells and promoting their in situ lysis to deliver BD antigens or DNA vaccine constructs into these host cells. Replication-incompetent HSV vectors engineered to express BD antigens will be studied in various cell lines for kinetics and levels of BD antigen expression. All BD antigen-expressing vaccines will be evaluated for their ability to induce neutralizing antibodies as well as CD8 + T-cell responses in mice. The vectors will also be tested for their ability to protect animals against anthrax toxin and/or bacterial and viral challenge. The most promising candidates will be evaluated for further development. This proj ect will use NERCE cores in Proteomics, Biological Molecule Production, and Animal and Clinical Testing (the latter, only after approval of human testing by IRB and appropriate committees).